According to the World Health Organization (WHO), around 17,000,000 people died of cardiovascular diseases in 2008. Thus, cardiovascular diseases are the most frequent cause of death among non-infectious diseases and are responsible for approximately one-third of all deaths worldwide. According to estimates, this number will rise to about 23,000,000 deaths per year by 2030.
Thus, cardiovascular diseases are not only the main cause of death worldwide but also cause enormous medical costs for national health systems and health insurance funds. Two of the most common and most damaging manifestations of cardiovascular disease are the occurrence of arteriosclerosis and thrombosis, which in return are, among other, responsible for heart attacks and strokes.
In recent years, major advances have been made in the treatment of cardiovascular diseases. This progress has been made possible not only by growing insights regarding the disease-inducing mechanisms, but also by the early identification of risk patients. In fact, the identification of disease risks and their early treatment are important characteristics of modern medical practice. Over the past 25 years, a variety of factors and clinical parameters have been identified that correlate either with the current condition of the disease or with the likelihood of cardiovascular disease in the future. Such risk factors can be measurable biochemical or physiological parameters such as, serum cholesterol, HDL, LDL and fibrinogen levels, but also behavioral patterns such as overweight and smoking. In cases in which a risk factor is not merely indicative of a disease or its development, but is actually causally involved in its development, a therapeutic influence on this risk factor can influence the course of the disease or reduce the risk of its development.
C-reactive protein (CRP) as an acute phase protein is part of the innate immune system and is formed in the liver in the course of inflammatory reactions and released into the blood. The formation of CRP is primarily induced by cytokines that are expressed in the context of an acute or chronic inflammatory reaction. The most powerful stimulus for the formation of CRP is the interleukin-6 (IL-6). Therefore, the levels of CRP as well as IL-6 in blood are indicators of a local or systemic inflammatory response. It is presumed that chronic inflammation is one of the underlying and supporting pathological manifestations of cardiovascular diseases. It is increasingly assumed that CRP is not only predicative for cardiovascular disease, but is also causally involved in its development or can influence its course.
Yeh (Clin Cardiol., 2005, 28, 408-412) shows that the CRP level can be used to predict cardiovascular disease risk, CRP is also an indicator of inflammatory responses, and that inflammation promotes all stages of atherosclerosis. Zoccali et al., (Semin. Nephrol. 2005, 25, 358-362) show that the CRP level is predictive of the cardiovascular mortality risk in patients with renal disease in the terminal stage. According to Nurmohamed et al., (Neth. J. Med. 2005, 63, 376-381), the CRP level is predictive of cardiovascular mortality risk in hemodialysis patients.
Sola et al. (J. Card. Fail. 2005, 11, 607-612) have shown that statin therapies can be used to reduce the amount of CRP and thus reduce the mortality and morbidity caused by cardiovascular disease. However, this form of therapy does not significantly reduce the high CRP levels (up to 1000-fold above normal values) resulting from a myocardial infarction or the high CRP levels in the blood of dialysis patients.
The normal value for CRP in the blood of humans varies from person to person, but is in the median at approximately 0.8 mg CRP per liter of blood, but can be in the case of acute or chronic inflammatory reactions (e.g. bacterial infections, atherosclerosis, after a heart attack) to levels substantially above 100 mg CRP per liter of blood. Since the half-life of CRP in the blood (approx. 19 hours) is constant and thus independent of the health condition of the patient, the synthesis rate of CRP alone is responsible for the regulation of the CRP level in the blood (Pepys & Hirschfield, J. Clin. Invest. 2003, 11, 1805-1812). Therefore, the greatly increased synthesis of CRP in acute pathological conditions places particular demands on therapeutic approaches to CRP removal of patients (risk patients or acute patients) since a significant amount of CRP needs to be removed in order to lower CRP levels to standard blood levels.
Consequently, there is an increasing interest in therapeutic procedures for reducing CRP levels in patients' blood. Due to the clinical significance of CRP there is also an interest in effective methods for the purification of CRP, in order to use the purified CRP in further experiments, e.g. for the investigation of its molecular function subsequently.
WO 2004/076486 discloses a method for inhibiting immunological, inflammatory and/or pathophysiological responses by administering CRP-binding molecules to patients with an increased CRP level. However, the extracorporeal treatment of biological fluids for the removal of CRP from said biological fluids is not disclosed.
WO 90/12632 discloses a method and device for the extracorporeal treatment of biological fluids with the aim of removing CRP as well as anti-phosphocholine antibodies from these biological fluids for the treatment of cancer. The phosphocholine-containing matrix used for this purpose can consist of e.g. silica, sepharose, acrylic beads or agarose, wherein both CRP and anti-phosphocholine antibodies are bound by means of the containing phosphocholine.
US 2007/225226 A1 discloses specific peptides which bind CRP and can therefore be used as a functional column material in a CRP apheresis. This binding takes place independently of calcium. Also some extracorporeal blood purification devices employing this column material for CRP apheresis are described, including a system for removing endotoxins using citrate as an anti-coagulant. CRP is a pentamer whose subunits are each associated with two Ca2+ ions which bind to the ligands used in this invention. Since citrate binds Ca2+ ions with high affinity, it has so far been assumed that when Ca2+-dependent ligands are used, citrate has a negative effect on the capacity of the columns to bind CRP. However, the inventors of the present application have shown that the capacity is higher for the used ligands. CRP is an opsonin and can activate the complement system via C1q in bound form. Surprisingly, however, it has been shown that citrate prevents the activation of the complement, which further increases the capacity so that citrate increases further the capacity of the used ligands. This effect occurring with Ca2+-dependent ligands is also completely surprising in the light of US 2007/225226 A1.
WO 2007/076844 discloses a method by extracorporeal CRP removal from the blood plasma by means of apheresis to reduce the risk to a patient (caused by an increased CRP level in the blood). According to the invention, a matrix-containing column is used to which phosphocholine derivatives are bound to bind CRP and remove it from the plasma, thus treating and/or preventing autoimmune diseases, cardiovascular diseases, diabetes and renal insufficiency.
Slagman et al. (Blood Purif. 2011, 31, 9) demonstrates on the pig model the successful reduction of CRP levels in the blood by extracorporeal apheresis after a heart attack.
A problem that occurs in the extracorporeal use of blood and blood plasma, whether in analytical or preparative applications but also in dialysis or apheresis, is the beginning of blood clotting, which hinder both the use itself or makes it impossible, but it also clogs as well as contaminates the used devices. For this reason, anti-clotting agents (so-called anticoagulants) are directly added to the blood or blood plasma after removal from the human or animal body.
One possibility for inhibiting blood coagulation is the administration of Ca2+ chelators such as EDTA (ethylenediaminetetraacetic acid), oxalate and citrates. Calcium is needed as a cofactor of some coagulation factors during blood clotting. Ca2+ chelators are used to remove calcium from the blood or blood plasma, thus inhibiting the coagulation. However, this is a significant problem for affinity chromatographic methods for the removal of CRP by means of phosphocholine or phosphoethanolamine or phosphocholine derivatives or phosphoethanolamine derivatives. Since the binding of CRP to phosphocholine or its derivatives Ca2+ is required (Black et al., Journal of Biological Chemistry, 2004, 279: 48487, Thompson et al., Structure 1999, 7 (2), 169-177), the unanimous opinion in the prior art was that an administration of Ca2+ chelators during the binding of CRP to phosphocholine should be avoided absolutely (see, for example, WO 90/12632). Thus, according to the prior art, the use of citrate-containing binding buffers during the affinity-chromatographic binding of CRP to phosphocholine is completely unsuitable. However, citrate-containing solutions (similar to administration of EDTA) were suitable as elution buffers, i.e. to remove the bound CRP from phosphocholine.
One possibility for Ca2+ independent anticoagulation consists in the administration of heparin. Heparin is a endogenous substance that is produced by mast cells and has an inhibitory effect on the coagulation cascade. By binding heparin to antithrombin II, a protease inhibitor circulating in the blood, which can inactivate activated coagulation factors such as thrombin and factor Xa, a conformational change of the antithrombin II is triggered. This accelerates the antithrombin II-mediated inactivation of coagulation factors by 2,000 times. Blood clotting comes to a standstill. Accordingly, in methods for the affinity-chromatographic removal of CRP from blood or blood plasma by means of phosphocholine and phosphocholine derivatives or phosphoethanolamine and phosphoethanolamine derivatives, heparin has hitherto exclusively been used as an anticoagulant.
However, this has some disadvantages, since heparin, especially in the case of unfractionated heparin, can lead to an immune reaction, the heparin-induced thrombocytopenia (HIT), in addition to hemorrhage. Thereby, heparin induces venous and arterial thromboses. In stroke patients in whom the use of CRP apheresis is also possible, the risk of HIT when receiving unfractionated heparin is approx. 3%. In addition, heparin is not approved for use with centrifugal cell separators which are often used to separate blood into the cellular components and the blood plasma.
It is an objective of the present invention to provide an anticoagulant for an affinity-chromatographic removal of CRP, which avoids the heparin-related problems from the state of the art and additionally does not impair the Ca2+-dependent binding of CRP to phosphocholine or phosphoethanolamine and their derivatives.
This objective is achieved by the teachings of the independent claims. Further advantageous embodiments are evident from the description, the examples and the dependent claims.
Surprisingly, it could be found that the use of a citrate solution for affinity-chromatographic removal of CRP from biological fluids using a column material functionalized with ω-phosphonooxyalkyl ammonium groups and/or with ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups not only surprisingly achieves an efficient removal of CRP from biological fluids but also solves the problems of the prior art.